Additional Glance on the Role of Dientamoeba fragilis & Blastocystis hominis in Patients with Irritable Bowel Syndrome.

Background
Irritable bowel syndrome (IBS) is a functional gastrointestinal disease with high population prevalence. Dientamoeba fragilis and Blastocystis hominis are reported worldwide as a cause of human gastrointestinal symptoms. This study evaluated the possible link between this syndrome and the infection with D. fragilis and B. hominis in Egypt.


Methods
Overall, 310 stool samples (160 from IBS patients and 150 from controls) were obtained from Tropical Medicine Outpatient Clinic, Faculty of Medicine, Ain Shams University, Cairo, Egypt between Mar 2015 and Feb 2016. All the stool specimens underwent direct examination and Trichrome staining. Each sample was cultivated on Jones and Robinson's media.


Results
Overall, 42 cases (28%) showed B. hominis and 2 cases (1.3%) for D. fragilis infections. After performing the culture methods for B. hominis and D. fragilis, detections increased to 50 cases (33.3%) and 3 cases (2%), respectively. While among 150 controls 18 (12%) positive samples were detected as B. hominis.


Conclusion
There may be a possible relationship between the presentation of irritable bowel syndrome and D. fragilis and B. hominis infections, which have to be excluded first.


Introduction
rritable bowel syndrome (IBS) is a functional gastrointestinal disease manifested by nausea, vomiting, and diarrhea usually is described as small volumes of loose stool preceded by urgency, frequent defeca-tion, diffuse abdominal pain, distention, and alternation in bowel habits in absence of a specific organic pathology. Several global studies recorded the prevalence of IBS to fall between 10%-20% (1). Diagnosis of Irritable Bowel Syndrome relies on the exclusion of other infections or bowel diseases; however, the most important diagnostic factor is the fulfillment of ROME III criteria (2). The therapeutic treatment targets the relief of symptoms with addition of psychotherapy (3).
Dientamoeba fragilis is a human intestinal protozoan parasite, commonly reported worldwide and associated with gastrointestinal manifestations (4). Firstly, mistaken by Jepps and Dobell who classified it as an amoeba (5). Then it is a trichomonad without flagella by molecular studies and electron microscopy (6). Dientamoeba remains neglected despite many reports of its pathogenic potential. This is due to the lack of studies on Dientamoeba compared to other intestinal protozoa such as Entamoeba histolytica and Giardia lamblia. Therefore, to say that D. fragilis is the exact cause of gastrointestinal disturbance, there must be no other pathogen detected (7).
Blastocystis hominis is a cosmopolitan human intestinal protozoan parasite (8). It is an anaerobic intestinal parasite of humans and animals. Moreover, B. hominis is the most common parasite found in human stool samples (9). Its prevalence rates are variable among countries (10). Prevalence is higher in developing (11,12) than industrialized (13,14) countries due to poor personal hygiene and consumption of contaminated food and water (15). The pathogenic ability of Blastocystis was widely discussed in different studies to determine whether this parasite was pathogenic or not (16). However, in-vitro and in-vivo studies showed that Blastocystis infection might be associated with gastrointestinal symptoms especially in patients with irritable bowel syndrome (17). The most common symptoms associated with Blastocystis infection include abdominal pain, diarrhea, and vomiting. There are many reports of patients that showed no other cause of gastrointestinal symptoms other than Blastocystis (18). Several studies associated Blastocystis with cutaneous manifestations in addition to gastrointestinal symptoms (19).
The methods commonly used to diagnose parasitic infections have a poor yield due to lack of experience in stool examination under microscopy, so staining methods and specific techniques such as cultures are needed to improve performance (29).
Newer molecular techniques, real-time PCR (qPCR) with multiplexing targeting the smallsubunit (SSU) ribosomal DNA (rDNA), have been developed and very attractive to be used (30). "The implementation of multiplex assays had a tremendous impact on routine parasitology laboratory studies in the developed world. However, the cost of such real-time PCR, assay in developing countries at present would prevent their application to routine diagnosis" (31).
The aim of this work was to evaluate for the first time in Egypt the possible link between patients with irritable bowel syndrome and the infection with D. fragilis and B. hominis.

Materials and Methods
This descriptive case-control study in which 310 stool samples were obtained from Tropical Medicine Outpatient Clinic, Faculty of Medicine, Ain Shams University, Cairo, Egypt between Mar 2015 and Feb 2016. The patients were fulfilling ROME III criteria of IBS (160) and the other 150 were controls. The controls were composed of healthy volunteers. The age of the participants ranged between 16-50 yr from both sexes.
These patients underwent thorough history and physical examination. All the stool specimens were processed by fresh direct film microscopy and another examination after stain-ing. Stool culture was done for B. hominis and D. fragilis.

Culture
For culturing B. hominis, Jones medium without starch was used (36). The cultures were incubated at 37 °C and examined after 48 h. If no B. hominis were seen up to further 2 d, they were regarded as negative. The sediment was examined under both the low power (×10) and high dry (×40) objectives. Robinson's medium was used to culture D. fragilis according to Windsor et al (37).

Statistical Analysis
The collected data was revised, coded, tabulated and introduced to a PC using SPSS. Data were analyzed using SPSS package ver. 15 (Inc., Chicago, IL). Chi-square test (Fisher's exact test) was used to examine the relation between qualitative variables. For quantitative data, comparison between two groups was done using Mann-Whitney test (nonparametric t-test).

Ethical consideration
An informed consent was taken from all patients before taking stool samples. The study was approved by the Research Ethics Committee, Faculty of Medicine, Ain Shams University.

Results
From the 150 patients presenting with IBS according to ROME III classification, 42 cases (28%) showed B. hominis (Fig. 1), 21 cases (14%) showed Entamoeba coli and 2 cases (1.3%) for each D. fragilis and Chilomastix mesnili infections. These results were shown after performing Trichrome staining technique. A note was made of the presence of other parasites such as Giardia intestinalis and Cryptosporidium spp., and these patients, ten in number, were excluded. No results revealed Enterobius vermicularis infection.
However, after performing the culture methods for B. hominis and D. fragilis detections increased to 50 cases (33.3%) and 3 cases (2%), respectively (Table 1). Comparison between cases and controls regarding B. hominis and D. fragilis as detected by microscopy and culture methods among cases and controls exhibited high significance in B. hominis cases (P=0.0001) and non-significant value (P=0.08) as regards D. fragilis cases. Moreover, the detected positive samples for B. hominis and D. fragilis among the 150 controls are 18 (12%) and zero (0%) respectively (Table 2).  Distribution of B. hominis and D. fragilis positive cases among their age groups was shown in Table 3 and Fig. 2. B. hominis positive cases were 8 (16%) under age of 20 yr, 21 (42%) from 20 to 30, 12 (24%) from 31 to 40 and 9 (18%) from 40 to 50 yr old. While for the D. fragilis positive cases, 2 (66.7%) cases were recorded from 20 to 30 aged group and only one case from 30 to 40 yr old. This may give a correlation of the most affected age group by both parasites which is the 20-30 age groups.

Discussion
There is a possible relationship between IBS and B. hominis and D. fragilis infections especially B. hominis infection (33.3%).
This coincides was investigated the prevalence of Dientamoeba and Blastocystis in irritable bowel syndrome patients (IBS) in Turkey (38). They did not find any stool sample positive for Dientamoeba but reported that many patients with IBS were found to have five or more Blastocystis parasites per field than control groups and they also reported regression of IBS symptoms after treatment in most of the patients with Blastocystis and lastly concluded that there may be a possible link between IBS and Blastocystis.
Moreover, Dientamoeba in 7.6% of fecal specimens from patients with enteritis was detected (39) and Dientamoeba in 0.9% of patients with diarrhea not detected in a control group of 900 patients without bowel complaints (40). Other study detected Dientamoeba in 4.5% of patients suffering from non-specific bowel disorders and 2% of patients with diarrhea (41). Dientamoeba was detected in 5.4% of patients with bowel complaints using light microscopy in combination with culture, PCR and real-time PCR (23).
Moreover, a study that aimed to screen for Blastocystis in colonic stool aspirate samples in patients with and without IBS undergoing colonoscopy and measured the interleukin levels (IL-3, IL-5, and IL-8) (42). They used stool cultures, polymerase chain reaction (PCR) for detection and subtyping of Blastocystis. Patients with IBS infected with only Blastocystis showed an increase in the interleukin levels which suggested that Blastocystis does have an effect on the immune system of the patient.
As an opposite opinion, a study was utilized a population-based sampling frame and Rome criteria assessment to examine the phenomenon of IBS in a "non-sterile" developing population, where repeated exposure to gastrointestinal pathogens is common and found B. hominis infection to be more in controls than IBS patients and stated a lack of an association between IBS and parasite infection in the developing nation environment of Nicaragua, Central America (43).
Although molecular methods specially Realtime PCR (qPCR) considered as an attractive technique for laboratory diagnosis of infectious diseases and would give more accurate results with D. fragilis (44), this could not be applied in the study due to high expenses and being not a routine method in developing countries.
Consequently, culture method was used as additional and confirmatory technique with the direct and staining as its effectiveness was proved over microscopy (29,36) and in sometime over the PCR (29).

Conclusion
There may be a possible relationship between Irritable Bowel Syndrome and D. fragilis and B. hominis infections, especially B. hominis infection.

Financial support
There has been no significant financial support for this work that could have influenced its outcome.